Calcein-AM (Calcein acetoxymethyl ester) is a cell-permeable green fluorescent probe commonly used for assessing eukaryotic cell viability or mitochondrial permeability transition pore, with no fluorescence itself. After hydrolysis, it emits green fluorescence with excitation/emission wavelengths: 494/515 nm.

The calcein-AM dye used to stain the living cells have a low spontaneous leakage rate of less than 15% in 4 hours at 37°C. Dilutions of targets stained by calcein-AM have a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs are readily detectable by this micro-test. Quantitation of killing and kinetic analysis is readily performed with the test system [1]. Calcein-AM is pH-independent, better retained, and more photostable. In addition, the high level of intracellular retention of calcein-AM and its low-level release after incorporation exclude possible cell-monolayer labeling and allow its use in a cell-cell interaction assay. Moreover, the bright fluorescence can easily be detected and measured by a microplate fluorescence reader [2]. Calcein-AM is a highly lipophilic vital dye that rapidly enters viable cells, is converted by intracellular esterases to calcein that produces an intense green (530-nm) signal, and is retained by cells with the intact plasma membrane. The calcein-AM assay has been used to assess cell viability, cytotoxicity, and tp quantitate apoptosis [3].